Mitochondrial dynamics and autophagy aid in removal of persistent mitochondrial DNA damage in Caenorhabditis elegans

Mitochondria lack the ability to repair certain helix-distorting lesions that are induced at high levels in mitochondrial DNA (mtDNA) by important environmental genotoxins and endogenous metabolites. These lesions are irreparable and persistent in the short term, but their long-term fate is unknown. We report that removal of such mtDNA damage is detectable by 48 h in Caenorhabditis elegans, and requires mitochondrial fusion, fission and autophagy, providing genetic evidence for a novel mtDNA damage removal pathway. Furthermore, mutations in genes involved in these processes as well as pharmacological inhibition of autophagy exacerbated mtDNA damage-mediated larval arrest, illustrating the in vivo relevance of removal of persistent mtDNA damage. Mutations in genes in these pathways exist in the human population, demonstrating the potential for important gene-environment interactions affecting mitochondrial health after genotoxin exposure.     

Genetic analysis shows that Rubus vikensis is a distinct species with a disjunct distribution

Rubus vikensis A. Pedersen ex G. Wendt (sect. Corylifolii) was recently described from a restricted area in north western Scania, Sweden. In this investigation, I show that the same species occurs also on the Onsala peninsula in northern Halland and on a single locality in the middle of Halland. It has 35 chromosomes in all parts of the distribution area. Moreover, I show by random amplified polymorphic DNA (RAPD) analysis that R. vikensis is a distinct and well‐defined species, clearly separated from the morphologically similar R. wahlbergii, with which it shares the chromosome number.     

The influence of axial ligands on the reduction potential of blue copper proteins

 The reduction potentials of blue copper sites vary between 180 and about 1000 mV. It has been suggested that the reason for this variation is that the proteins constrain the distance between the copper ion and its axial ligands to different values. We have tested this suggestion by performing density functional B3LYP calculations on realistic models of the blue copper proteins, including solvent effects by the polarizable continuum method. Constraining the Cu-S_Met bond length to values between 245 and 310 pm (the range encountered in crystal structures) change the reduction potential by less than 70 mV. Similarly, we have studied five typical blue copper proteins spanning the whole range of reduction potentials: stellacyanin, plastocyanin, azurin, rusticyanin, and ceruloplasmin. These studies included the methionine (or glutamine) ligand as well as the back-bone carbonyl oxygen group that is a ligand in azurin and is found at larger distances in the other proteins. The active-site models of these proteins show a variation in the reduction potential of about 140 mV, i.e., only a minor part of the range observed experimentally (800 mV). Consequently, we can conclude that the axial ligands have a small influence on the reduction potentials of the blue copper proteins. Instead, the large variation in the reduction potentials seems to arise mainly from variations in the solvent accessibility of the copper site and in the orientation of protein dipoles around the copper site. Received: 7 April 1999 / Accepted: 26 July 1999     

Which functional groups of the molybdopterin ligand should be considered when modeling the active sites of the molybdenum and tungsten cofactors? A density functional theory study

A density functional theory study of the influence of the various functional groups of the molybdopterin ligand on electronic and geometric properties of active-site models for the molybdenum and tungsten cofactors has been undertaken. We used analogous molybdenum and tungsten complexes with increasingly accurate representation of the molybdopterin ligands and compared bond lengths, angles, charge distribution, composition of the binding orbitals, as well as the redox potentials in relation to each other. On the basis of our findings, we suggest using ligand systems including the pyrane and the pyrazine rings, besides the dithiolene function, to obtain sufficiently reliable computational, but also synthetic, models for the molybdenum and tungsten cofactors, whereas the second ring of the pterin might be neglected for efficiency reasons.     

Increased therapeutic effect of vinca alkaloids targeted to tumour by a hybrid-hybrid monoclonal antibody

Summary Unmodified vinblastine (VLB) targeted through one of the antigen combining sites of the hybrid-hybrid 28.19.8 monoclonal is potentially more effective in suppressing the growth of established MAWI tumour xenografts implanted on nude mice than free VLB in the absence of the targeting agent, presumably due to an increased local drug concentration. Our efficacy results in this study suggest that drug, specifically removed from the circulation by hybrid-hybrid antibody previously located to the tumour mass, can be made available in a pharmacologically active from. Histological analysis of the treated tumours revealed dramatic changes in the tumour organisation with only a few surviving tumour cells with altered morphology.     

Measurements of total body calcium by prompt-gamma neutron activation abalysis using a252Cf source

A clinical neutron activation instrument has been developed for in vivo elemental analysis. Utilizing the prompt-capture gamma ray technique, simultaneous total body (TB) measurements of primarily Ca, but also Cl, N, C, and H are routinely performed. This paper describes a technique for the measurement of TBCa (g) that relies on the use of TBCl as an internal standard. The method has been tested with four anthropomorphic phantoms covering a range of body habitus. The mean discrepancy between the measured and known Ca contents was 3.6%. The technique has been applied to two patient groups, and encouraging results were obtained.     

Fine Structural Observations on the Extracellular Matrix (ECM) of Xenoturbella bocki Westblad, 1949

Fine structural observations on the extracellular matrix (ECM) and connective tissue system of the enigmatic vermiform animal Xenoturbella bocki have demonstrated a complex and interesting organization of the ECM. Most conspicuous is the subepidermal membrane complex (SMC), and the major part of the ECM is present in this structure, which consists of a limiting basal lamina on each side of a central thick filamentous layer, probably of a collagenous nature. Distinct anchoring filaments are found as part of the SMC. A basal lamina is also observed in connection with the gastrodermal cells. The mesoderm is represented by a loose, ill-defined parenchyma, without filaments. The SMC is discussed in relation to subepidermal basal matrices occurring in turbellarians and enteropneusts, the two groups to which a relationship to Xenoturbella previously has been suggested. Comparisons between the ECM in Xenoturbella and in turbellarians do not support the notion of a relationship between these groups. Conversely, the present study strengthens previous indications of distinct similarities between certain characteristics of the epidermis and the SMC present in both enteropneusts and in Xenoturbella.     

Protonation states of intermediates in the reaction mechanism of [NiFe] hydrogenase studied by computational methods

The [NiFe] hydrogenases catalyse the reversible conversion of H₂ to protons and electrons. The active site consists of a Fe ion with one carbon monoxide, two cyanide, and two cysteine (Cys) ligands. The latter two bridge to a Ni ion, which has two additional terminal Cys ligands. It has been suggested that one of the Cys residues is protonated during the reaction mechanism. We have used combined quantum mechanical and molecular mechanics (QM/MM) geometry optimisations, large QM calculations with 817 atoms, and QM/MM free energy simulations, using the TPSS and B3LYP methods with basis sets extrapolated to the quadruple zeta level to determine which of the four Cys residues is more favourable to protonate for four putative states in the reaction mechanism, Ni-SI_a, Ni-R, Ni-C, and Ni-L. The calculations show that for all states, the terminal Cys-546 residue is most easily protonated by 14–51 kJ/mol, owing to a more favourable hydrogen-bond pattern around this residue in the protein.     

Redesign of the coenzyme specificity in L-lactate dehydrogenase from bacillus stearothermophilus using site-directed mutagenesis and media engineering.

L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox enzyme which has a strong preference for NADH over NADPH as coenzyme. To exclude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspartate residue at position 52. However, this residue is probably not solely responsible for the NADH specificity. In this report we examine the possibilities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, after choosing the mutant with the highest selectivity for NADPH, we also investigated the possibility of further altering the coenzyme specificity by adding an organic solvent to the reaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turnover number of this mutant was increased almost fourfold as compared with wild-type LDH. To explain the altered coenzyme specificity exhibited by the D52SI51K double mutant, molecular dynamics simulations were performed.